Cytosolic Ca2+ is a critical second messenger in virtually every type of cell. Ca2+ is novel relative to other messenger molecules because it regulates multiple processes simultaneously within an individual cell. The ongoing theme of this research project is to investigate how Ca2+ regulates bile secretion in hepatocytes in particular, as a model for how Ca2+ can regulate a localized process within a polarized epithelium. In the hepatocyte, Ca2+ signals are mediated entirely by inositol 1,4,5-trisphosphate (InsPS). InsPS acts by binding to the InsPS receptor (InsPSR), which is a tetrameric InsPS-gated Ca2+ release channel in the endoplasmic reticulum (ER) membrane. InsPS thus increases cytosolic Ca2+ by releasing Ca2+ from the ER via the InsPSR. Two of the three known isoforms of the InsPSR (lnsP3R-1 and lnsP3R-2) are expressed in hepatocytes, each with distinct biophysical properties. Each of these two isoforms is distributed in a distinctive subcellular pattern in the hepatocyte, which thus may establish signaling microdomains within the cell. The hypothesis of this proposal is that Ca2+ signaling patterns and their effects on hepatocyte bile secretion depend upon the types of InsPSRs that are expressed and their subcellular distributions. This hypothesis will be tested through three specific aims: (1) The relative effects of lnsP3R-1 and lnsP3R-2 on Ca2+ signaling will be determined at the molecular and single-channel level; (2) The relative effects of lnsP3R-1 and lnsP3R-2 on Ca2+ signaling will be determined in intact cells; (3) The relative effects of lnsP3R-1 and lnsP3R-2 on Ca2+-mediated secretion will be determined in model cell systems and in the intact, perfused liver. Together, these studies will provide new insights to the molecular and cellular basis for cholestasis, one of the cardinal manifestations of liver disease. Moreover, this work should provide a general paradigm for the way in which Ca2+ signals are generated in order to regulate events within specific subcellular regions.